International Journal of Micro Biology, Genetics and Monocular Biology Research
Vol.2, No.2, pp.9-20, June 2016
___Published by European Centre for Research Training and Development UK (www.eajournals.org)
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RAPID IDENTIFICATION OF ENTEROBACTER SPP. ISLATED FROM
HOSPITALS IN BASRAH PROVINCE BY AUTOMATED SYSTEM (VITEK®2
COMPACT)
Prof.Yahya A. Abbas1 and Ghosoon Fadhel Radhi2
1Nassiriya Tech.Institute.Southern Tech.University 2Department of Biology, College of Science,University of Basrah,Iraq.
ABSTRACT: Atotal of 676 samples were taken from various hospitals in Basrah province.
These included clinical specimens(urine , blood , stool ,nasal swabs, throat swabs,ear
swabs),Environmental swabs(beds,tables,ground)and milk powder of children.All isolates
were subjected to the cultural,microscopical,biochemical examination and vitek2 compact
used for identification of bacteria.Atotal of 153 bacterial isolates were diagnosed as
Enterobacter(67 isolates E.aerogenes,65 isolates E.cloacae complex,11 isolates E.cloacae
subsp cloacae ,4 isolates E.cloacae subsp dissolvens,4 isolates E.sakazakii,1 isolate
E.hormaechei and 1 isolate E.asburiae) .
KEYWORDS: Enterobacter, Vitek®2 Compact, Basrah Province
INTRODUCTION
Enterobacter belongs to domain bacteria, phylum proteobacteria, class gamma-prteobacteria,
order enterobacteriales family enterobacteriaceae (Brenner etal., 2004). Enterobacter was first
described by Hormaeche and Edwards(1960). Enterobacter are rod-shaped cells,motile by
peritrichous flagella,some of which are encapsulated.All Enterobacter spp. facultative
anaerobes and do not form spores(Mezzatesta etal., 2012).They are biochemically active and
ferment sugars such as glucose,arabinose,maltose,xylose,often with gas production. They are
oxidase negative,catalase positive and reduce nitrate to nitrite(Murray etal.,2003),Voges-
proskauer test is usually positive, Enterobacter species are ubiquitious and widely found in
nature these microorganisms are saprophytic in the environment and commensal in the enteric
flora since they are found in soil and sewage, as well as in the gastrointestinal tract of human
(Leclerc et al.,2001; Mezzatesta et al.,2012). It is diverse bacterial genus consisting of several
species like E. aerogenes and E. cloacae have been reported as important opportunistic
pathogens for human, These bacteria have been largely described during several outbreaks of
hospital-acquired infectious in Europe and particularly in france (Davin-Regli and pages,2015).
Enterobacter spp. can create community infections are responsible for approximately half of
all nosocomial acquired infections(Huang etal.,2001). Enterobacter has undergone numerous
taxonomical rearrangement.Study of Rezzonico etal. (2009)indicated that many strains
previously identified as E. agglomerans and have been transferred into the genus Pantoea
agglomerans. E.aerogenes is considered a homotypic synonym of Klebsiella mobilis because
it has the same type strain(Skerman etal.,1980). Previously reported that E. sakazakii was 53-
54% related to two genera Enterobacter and Citrobacter by DNA-DNA hybridization(Farmer
etal.,1980).E.sakazakii was placed in Enterobacter genus because of its closer phenotypic and
genotypic relationship to E. cloacae than to C,freundii.
International Journal of Micro Biology, Genetics and Monocular Biology Research
Vol.2, No.2, pp.9-20, June 2016
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In year 2007 and 2008,classification of E. sakazakii with the creation of new genus,
Cronobacter, has been proposed based on biotyping and genotyping studies (Iversen etal.,
2007; Iversen etal.,2008).
The routine identification of bacterial isolates in microbiology laboratory is currently done by
analysis of phenotypic features such as growth on selective and nonselective media ,colonial
morphology ,Gram-stain,biochemical reactions .These methods are laborious,time-consuming
(Darbandi,2010;Jamal.;2014).
The vitek 2 compact is an automated microbiology system utilizing growth based technology
and designed for the identification and susceptibility testing of wide range of micro organisms
including Gram-negative and Gram positive bacteria and yeasts in clinical or industrial
samples(Ling et al 2001;Darbandi,2010).This system use colorimetric reagent cards that are
incubated and interpreted automatically(Pincus.2005). Figure (1) shows the VITEK 2 compact.
The purpose of this study was to identify of Enterobacter spp. isolated from different areas by
using automated systemVitek®2 compact.
Fig(1):Vitek 2 compact Instrument
MATERIALS AND METHODS
Samples collection
Six hundred and sixsty seven samples were collected from different areas of Basrah hospitals
(Al-Fayhaa General hospital ,Al-Mawanee General hospital , Al-Sadder teaching hospital , Al-
Basrah hospital for gynecology and obstetrics , Al-Basrah childrenʼs specialty hospital, Al-
Basrah General hospital).The collected samples represent clinical and environmental samples
,Clinical specimen including blood, urine, stool, nasal swabs , throat swabs , ear swabs)
.Environmental swabs were taken from beds,tables,ground and food samples represented by
milk powder of children’patients suffering from diarrhea.All samples were collected under
sterile conditions and sent to the laboratory within 1-2 hrs.
Isolation and Identification of Bacteria.
Isolation from milk powder(FDA,2002)
One gram of milk powder of infants from each sample was mixed with 9ml sterile peptone
water.The tubes were then incubated for 24hrs at 37 °C, and 0.1ml of each suspension was
streaked on violet red bile agar (VRBA) the plates were incubated for24hrs at 37 °C ,red
colonies appeared on VRBA were subcultured by streaking on tryptic soya agar (TSA) and
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11
incubated for 24hrs at 25°C.The colonies that produce yellow pigment were identified using
traditional biochemical tests and vitek2 compact.
Isolation from other specimens.(Mano and Byku,2012)
All specimens were inoculated on various ordinary media; blood agar, MacConkey agar, EMB
agar and incubated at 37 °C for 24 hrs under aerobic conditions, after that the culture plates
were examined according to the appearance, color and morphology of the colonies and Positive
cultures were subjected to biochemical tests( sugar fermentation, IMVC,
TSI,Oxidase,Catalase) for identification of bacteria.
Confirmatory Identification of Enterobacter spp by Vitek® 2 compact
All isolated were cultured on nutrient agar and incubated for 24hrs at 37 °C to ensure purity
and to get single colonies ,after isolation of bacterial colonies on culture media,isolates were
identified by Vitek® 2 compact auto analyzer system manufactured by
(BioMerieux,USA)Public health lab. In Najaf and this process including several
steps(Pincus,2005;Darbandi,2010):
1-Asterile plastic stick applicator used to take pure colonies from culture media and transfer
sufficient number of them to clear plastic(polystyrene) test tube 12×75 mm contain about 3
ml of sterile saline(NaCl 0.45%-0.50%,pH=7)BioMerieux,USA to suspend the
microorganism in.
2-Concentration of bacterial suspension in saline was measured by a densitometer and adjusted
to 0.50-0.63 Mcfarland before introducing the sample to the analyzer.
3-The turbidity of bacterial suspension was adjusted by adding proper amount of bacteria or
normal saline and mixing by shaker to produce a homogenous suspension of bacteria.
4-The turbidity (the density) of the suspension was checked by using a calibrated turbidity
meter called the Densichek
5-Identification GN cards were loaded (inoculated) with bacterial suspension using an
integrated vacuum apparatus.
6-A test tube containing the bacterial suspension is placed into a special rack(cassette) and the
identification card is placed in the neighboring slot while inserting the transfer tube into the
corresponding suspension tube, the cassette contain place for 10 test tubes.
7-The filled cassette was placed into a vacuum chamber station inside the vitek® 2 compact
machine .
8- After the vacuum is applied and air is re-introduced into the station,the bacterial suspension
was forced through the transfer tube into micro-channels that filled all the test wells.
9- Inoculated GN cards were passed by a mechanism,which cut off the transfer tube and sealed
the card prior to loading into the circular incubator.
10-Circular incubator could accommodate up to 30 cards,all card types were incubated at
35.5+1 °C.
11-Each card was removed from the incubator every 15 minutes,transported to the optical
system for reaction readings, and then returned to the incubator until the next read time.
12-Data were collected at 15 minute intervals during the entire incubation period.
13- Vitek 2 compact tests listed in Table (1)
Table (1) Test Substrates of Vitek 2 compact
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12
Well
number
Mnemonic
Biochemical Test
2 APPA Ala-Phe-Pro-ARYLAMIDASE
3 ADO ADONITOL
4 PyrA L-Pynolydonyl-ARYLAMIDASE
5 IARL L-ARABITOL
7 dCEL D-CELLOBIOSE
9 BGAL BETA-GALACTOSIDASE
10 H2S H2SPRODUCTION
11 BNAG BETA-N-ACETYL-GLUCOSAMINIDASE
12 AGLTo Glutamyl Arylamidase pNA
13 dGLU D-GLUCOSE
14 GGT GAMMA-GLUTAMYL-TRANSFERAS
15 OFF FERMENTATION/GLUCOSE
17 BGLU BETA-GLUCOSIDASE
18 dMAL D-MALTOSE
19 dMAL D-MANNITOL
20 dMNB D-MANNOSE
21 BXYL BETA-XYLOSIDASE
22 BAlap BETA-Alanine arylamloase pNA
23 ProA L-Proline ARYLAMIDASE
26 LIP LIPASE
27 PLE PALATINOSE
29 TyrA Tyrosine ARYLAMIDASE
31 URE UREASE
32 dSOR D-SORBITOL
33 SAC SACCHAROSE/SUCROSE
34 dTAG D-TAGATOSE
35 dTRE D-TREHALOSE
36 CIT CITRATE(SODIUM)
37 MNT MALONATE
39 5KG 5-KETO D-CLUCONATE
40 ILATk L-LACTATE alkalinisation
41 AGLU ALPHA-GLUCOSIDASE
42 SUCT SUCCINATE alkalinisation
43 NAGA Beta-N-ACETYL-GALACTOSAMINIDASE
44 AGAL ALPHA-GALACTOSIDASE
45 PHOS PHOSPHATASE
46 GlyA Glycine ARYLAMIDASE
47 ODC ORNITHINE DECARBOXYLASE
48 LDC LYSINE DECARBOXYLASE
52 ODEC DECARBOXYLASE BASE
53 IHISa L-HISTIDINE assimilation
56 CMT COUMARATE
57 BGUR BETA-GLUCORONIDASE
58 O129R O/129RESISTANCE(comp.vibrio)
59 GGAA Glu-Gly-Arg-ARYLAMIDASE
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61 IMLTa L-MALATE assimilation
62 ELLM ELLMAN
64 ILATa L-LACTATE assimilation
RESULTS
A total of 676 Clinical , environmental and food specimens were collected during the
period(January 2013-December 2013),from Basrah hospitals,The clinical specimen included
blood(50) ,urine(100) ,stool (100), nasal ,throat and ear (50) and environmental specimens
included patient bed swabs (200) , tables swabs (100),patient room ground swabs (50) and food
specimens (milk powder of infants specimens ( 26)(Table ,2). 170 Enterobacter spp. were
identified during this study.
Table (2): Distribution Enterobacter spp. in various samples
Sample
No(%)0f
samples
Number Enterobacter spp.
Total
number of
Enterobacter
spp.
N % Clinical specimens
7.4%
EC. E.
aero
genes
E.
saka
zakii
E.
horma
echei
E.
asbu
riae
*EC1=1
0
0
0
0
1
0.7
Blood(n=50)
Urine(n=100) 14.79% EC1=1 4 0 0 0 5 3.3
Stool(n=100) 14.79% EC1=1
**EC2=1
4 0 0 0 6 3.9
Nasal,Throat,ear(n=50) 7.4% 0 0 0 0 0 0 0
Environmental specimens
29.59%
EC1=44
EC2=10
EC3=3
51
2
0
0
110
71.9
Patient bed(n=200)
Patient tables in different
hospitals(n=100)
14.79% EC1=16
***EC3=1
7 2 1 0 27 17.6
Patient room ground in
different hospitals(n=50)
7.4% EC1=2 0 0 0 0 2 1.3
Food specimens 3.8% 0 1 0 0 1 2 1.3
Milk powder
infant(n=26)
Total=153
*EC1=Enterobacter cloacae complex
**EC2=Enterobacter cloacae subsp cloacae
***EC3=Enterobacter cloacae subsp dissolvens
Identification and characterization of Enterobacter spp.
Characterization of Enterobacter spp. used in the present study was carried out in accordance
with conventional methods(Gram stain,morphological characterization and biochemical
tests)(Table,3) . All isolates were also confirmatory identified by using Vitek 2 compact GN
colorimetric card was read and interpreted automatically with Vitek 2 compact system.
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Table (3): Morphological and Biochemical tests which were used to identify Enterobacter
spp.
Result Biochemical test No.
- Gram stain 1
Rod Morphological shape 2
- Indole test 3
- Methyl red test 4
+ Voges-Proskauer test 5
+ Citrate utilization test 6
+/+,G TSI,B/S 7
+/- Gelatin hydrolysis 8
- Oxidase test 9
+ Catalase test 10
+ Motility test 11
V Aesculin hydrolyzed 12
V Utilization of raffinose 13
- DNase test 14
(+)=Positive,(-) =Negative,(V) =Variable,G=Gasis produced,B/S=Butt/Slant
TSI=Triple sugar iron
Identification of Enterobacter spp. by Vitek 2 compact
Identification of 170 Enterobacter species depending on biochemical reactions (Table ,3)were
confirmed by Vitek 2 compact .Vitek 2 Results showed that(153) isolates were identified as E.
species as following 65(42.5%)isolate were identified as E.cloacae complex, 11(7.2%)isolate
were identified as E.cloacae spp cloacae , 4(2.6%) isolate were identified as E.cloacae spp
dissolvens, 67(43.80)isolate were identified as E.aerogenes, 4(2.6%)isolate were identified as
E.sakazakii , 1(0.7%) isolate was identified as E.hormaechei, 1(0.7%) isolate was identified as
E.asburiae (Fig ,2). The other (17) isolates were unidentified by Vitek 2 compact. Among all
bacterial isolates obtained only 12(7.84%) Enterobacter spp were isolated from clinical
specimens and recognized as (3)E cloacae complex,(1)E. cloacae ssp cloacae,(8)E. aerogenes,
while 139(90.85%)isolates of Enterobacter spp were isolated from hospital environmental
swabs and recognized as(62)E. cloacae complex,(10) E.cloacae ssp. cloacae,(4)E.cloacae ssp.
dissolvens (58) E.aerogenes (4)E.sakazakii,(1)E.hormeachei .Also 2(1.31%)of Enterobacter
spp were isolated from infant milk powder and recognized as (1)E.aerogenes,(1)E.asburiae
Table(4).
The results revealed that the isolates identified at the species level was divided into four groups
based upon the probability of accurate identification as follows: 59.48% isolate were
excellent(probability of accurate identification(96-99%),28.10% isolate were very good(93t o
95%),11.76% isolate were good(89 to 92%) and 0.70% isolate acceptable(85 to 88%) as gave
in Fig(3).
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Figure (2)Percentage of Enterobacter spp.isolated from different samples by using vitek
2 compact
Table (4): Number of samples collected and Enterobacter spp. isolated from clinical ,
Environmental and food specimens
Fig(3):Probability of identification of Enterobacter spp. by Vitek 2 compact
Probability of excellent identification =96-99%
Probability of very good identification =93-95%
Probability of good identification =89-92%
Probability of Acceptable identification =85-88%
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
40.00%
45.00%
E.cloacae complex
E.cloacaessp cloacae
E.cloacae spp dissolvens
E.aerogenes
E.sakazakii
E.hormaechei
E.asburiae
(65)42.5%
(11)7.2%( (4)2.6%
(67)43.7%
(4)2.6%(1)0.7%(1)0.7%
59.48%28.10%
11.76%
0.70%Excellent Very good Good Acceptable
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DISCUSSION
Infections due to Gram negative bacteria has become an increasing problem in recent
years(Sankarankutty and Kaup,2014).The present study revealed among 676 samples
from(clinical , food and environment of Basrah hospitals) that153 were Enterobacter spp and
most bacterial isolates from hospital environments, The results showed that Enterobacter spp
were distributed as follow:139(90.85%)from hospital environmental
specimens,12(7.84%)from clinical specimens,2(1.31%)from milk powder specimens as
showed in Table(4),These results indicated wide distribution of Enterobacter spp in the
environment of hospitals and this agreement with other studies(Jalaluddin
etal.,1998;NNIS,2004;Chang etal.,2009).Enterobacter is more a nosocomial opportunistic
pathogen that cause variety of hospital acquired infections (Sanders and sanders,1997;Gupta
etal.,2003). National healthcare safety network reported that Enterobacter account for
approximately 5% of nosocomial bacteremia in 2008(Hidron etal.,2008).especially in
intensive care units(ICUs)(Boban etal.,2011).Hospital environments are responsible of the
dissemination of microorganism for different distances and progressive contamination of
surfaces,water and air (Boyce etal.,1997;Curtis,2008).The results showed that a higher
percentage(43.80%)of isolates were identified as E. aerogenes followed by E.cloacae
complex(42.5%),E.cloacae sub sp cloacae(7.2%),E.cloacae sub sp dissolvens and E.sakazakii
with the same percentage(2.6%),E.hormaechei and E.asburiae with the same
percentage(0.7%).These results indicated the most frequently E.aerogenes and E.cloacae
complex and less frequently E.hrmaechei and E.asburiae as reported previously(Yu et
al.,2000). Al-Tawfig et al.(2009) also proved that E.cloacae and E. aerogenes constituted 60%
and 33% of their isolates,respectively .
Hussain and Alammar(2013) recorded higher percent for E. cloacae (89.3%)from various
hospitals of Najaf/Iraq,on the other hand E. aerogenes is considered the fifth highest
Enterobacteriaceae and the seventh highest Gram negative Bacilli responsible for notorious
nosocomial infections in france(Carbonne et al.,2013).E.aerogenes and E.cloacae have been
largely described during several outbreaks of hospital acquired infection in Europe especially
in France because these bacterial species are able to acquire numerous genetic mobile elements
that contribute to antibiotic resistance,this help them to colonize several environments and host
and rapidly adapt their metabolism to external conditions and environmental stresses(Davin-
Regli and Pages,2015).
Identification of Enterobacter spp
Identification of E. spp depended on morphological ,microscopic examination and biochemical
tests. revealed that all Enterobacter isolates were gram negative and rod shape according to
results recorded in Bergey's manual of determinative bacteriology(1994) and Bergey's manual
of systematic bacteriology(2004) (Holt etal.,1994;Brenner etal.,2004).
Identification of Enterobacter spp by Vitek 2 compact
Identified isolates of Enterobacter by convensional methods were confirmed with the
automated vitek2compact system by using GN-ID cards.In this study 153 isolate identified to
species and subspecies level of Enterobacter this depended on difference in colorimetric
International Journal of Micro Biology, Genetics and Monocular Biology Research
Vol.2, No.2, pp.9-20, June 2016
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17
measurements that taken every 15 minutes by vitek 2 compact system for each isolate.Observed
during the present results some isolates had the same species and appeared differentce in some
biochemical tests this indicated they belong to different strains, strains identification at the
species level were divided into four groups as showed in fig(3) based on the probability of
accurate identification as follows excellent (probability of accurate identification ≥96%),very
good(93-95%),good(89-92%)and acceptable(85-88%)(Zbinden et al.,2007).Vitek 2 compact
system had several advantages ,it identified a significant number of Gram negative bacteria
during 6 hrs which clinically relevant,because rapid reporting of microbiology results
compared with traditional methods that require two or three days and has high level of
automation ,a simple methodology and taxonomically updated databases (Ling
etal.,2003;Wallet et al.,2005;Otto-Karg et al.,2009;Dina et al.,2014).Vitek 2 compact
incorporates several technical improvements which automate many procedures that performed
manually with the previous vitek system(Decueto et al.,2004).
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